1052
J. Phys. Chem. 1988, 92, 1052-1055
Extended 2,5-~enyloxazale-y-CycCodextrin Aggregates EmMtng 2,5-Diphenyloxazole Excimer Fluorescence Rezik A. Agbariat and David.Gill* Department of Physics, Ben Gurion University of the Negev, Beer Sheva 84105, Israel (Received: June 12, 1987)
2,5-Diphenyloxazole(PPO), a common scintillator and laser dye, forms inclusion complexes with cyclodextrins (CD) in water. We prepared highly concentrated complex solutions, of which only P m - C D gave rise to extended aggregates. Their properties are as follows: (1) visible turbidity; (2) PPO excimer fluorescence, emitted only by these aggregates; (3) high polarization of excimer fluorescence (P = 0.25), yielding an aggregation number N 60; (4) laser scattering, yielding N 500. The aggregate is believed to be a coaxial array of 7-CD beads, linked by overlapping pairs of PPO molecules. N
The watec-soluble cyclic oligosaccharides CY-, fi-, and y-cyclodextrins (CD; cycloamyloses) can form inclusion complexes with guest molecules.'*2 y-CD molecules are shaped as tube sections of 0.7 nm length, 1.69 nm o.d., and 0.85 nm i.d. The cavities of these digestible molecules can encapsulate labile or distasteful drugs and this application is being introduced on a large scale.2 Various influences of C D inclusion complexing on guest molecules have been reported: (1) sheltering of the hidden parts3v4and enhanced reactivity of the exposed partsS of the guest molecules; (2) strong interactions within a guest pair cag'ed in a y-CD cavity,@ and (3) self-assembly of extended molecular arrays, held together by inclusion c ~ m p l e x i n g . ~ We wish to report an outstanding case, where pair interaction (phenomenon 2) was made possible only as a consequence of macroaggregation (phenomenon 3). 2,5-Diphenyloxazole (PPO, Figure l ) , a common scintillator and laser dye,I0 was known to emit weak excimer fluorescence in concentrated solutions.1h12 This fluorescence had a surprisingly long lifetime of 13.6 ns, whereas monomer lifetime in solution was only 1.6 ns.13-16We planned to insert two PPO molecules in a single y-CD cavity and thus enhance the excimer yield. Contrary to this expectation, excimer emission appeared only in extended PPO-y-CD aggregates.
Materials and Methods Cyclodextrins and PPO were purchased from Aldrich and were used without further purification. Fluorescence grade ethanol and cyclohexane, as well as triply distilled water, were used. The problem of the quantitative insertion of hydrophobic guests into C D had been faced by many w o r k e r ~ . ~Solubilizing ~J~ agents may penetrate the C D cavities and hence should be avoided. Alternatively, when hydrophobic compounds are directly applied to water, they may disperse into microcrystal suspensions. An embarassing situation is encountered when suspended crystallite spectra are indistinguishable from the spectra of CD inclusion complexes.20 In ref 19 the suggestion was made to prepare clear submicromolar solutions of the hydrophobic solute in water and then add the CD. We tried this approach, but found the low concentration to be restrictive and, besides, the total absence of suspended crystallites was hard to prove even then. We present a preparative method, which was absolutely essential for obtaining the reported results: (a) PPO was dissolved in ethanol at a concentration determined spectrophotometrically. (b) A measured volume of ethanol solution was allowed to wet the walls of the preparative test tube. (c) The solvent was vaporized, leaving adsorbed dye on the wall. (d) The water solution of CD was poured in and incubated for 2 h, so as to allow the sequestering of PPO from the wall. Silica gel powder, repeatedly wetted with PPO solution and dried, had larger carrying capacity than the glass walls.2' Good 'In partial fulfillment of the requirements for a MSc. thesis, Department of Physics, Faculty of Natural Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
-
practice would have included thorough drying in vacuo, which would eliminate any transfer of residual solvent into C D cavities. In our experience, even moderate drying of loaded silica gel resulted in good yields. The efficient preparation method has yet to be matched by an assay of CD-bound ligand. Assays based on nuclear magnetic resonance or ultraviolet absorption of complexed ligand did not respond well. Fluorescence assay would make sense only after the complexed ligand was extracted into a standard solvent. Heretofore, extraction attempts have produced quasi-stable emulsions. While an assay procedure was worked on, we had to adopt the following assumptions: (1) with solid-adsorbed ligand in excess, the amount of complexed ligand is proportional to added CD and (2) with C D in excess, complexed ligand is proportional to solid-adsorbed added ligand. Absorbance was measured on Bausch & Lomb Spectronic 2000. Steady fluorescence excitation, emission, and anisotropy were determined on a Perkin-Elmer MPF-44. These measurements were also applied to dry samples of the aggregate and of PPO powder. Phase and modulation lifetimes were taken on a SLM4800 phase fluorometer but were not analyzed in terms of polyphasic decays. Impulse response measurements were made on a time-correlated single-photon fluorometer which had a N 2 microflash. Nitrogen emissions at 316 and 358 nm coincided with respective M and D excitation peaks. Decay curves were deconvoluted by global analysis programs, kindly provided by (1) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; SpringerVerlag: West Berlin, 1978. (2) Bender, H. Adu. Biotechnol. Processes 1986, 6, 3 1-71. (3) Turro, N.; Bolt, J.; Kuroda, Y.; Tabushi, I. Photochem. Photobiol. 1982, 35, 69-72. (4) Scypinski,
S.;Cline Love, L. J. Int. Lab. 1984 (April), 60-64. (5) Breslow, R. Acc. Chem. Res. 1980, 13, 170-177. (6) Yorozu, T.; Hoshino, M.; Imamura, M. J. Phys. Chem. 1982, 86,
4426-4429. (7) Ueno, A.; Takahashi, K.; Osa, T. J. Chem. SOC.,Chem. Commun. 1980,921-922. (8) Kobayashi, N.; Ueno, A.; Osa, T. J. Chem. SOC.,Chem. Commun. 1981, 340. (9) Edwards, H. E.; Thomas, J. K. Carbohydr. Res. 1978,65, 173-182. (10) Berlman, I. B. Handbook offuorescence spectra of aromatic molecules; Academic: New York, 1971; pp 291-294. (11) Berlman, I. B. J . Chem. Phys. 1961, 34, 1083. (12) Birks, J. B.; Christophoru, L. G. Proc. R. SOC.London, A 1964,277, 571-582. (13) Birks, J. B.; Dyson, D. J.; Munro, I. H. Proc. R. SOC. London, A 1963, 275, 515-588. (14) Ware, W. R.; Watt, D.; Holmes, J. D. J. Am. Chem. Soc. 1974, 96, 7853-7864. (15) Birch, D. J. S.; Imhof, R. E.; Dutch, A. J. Lumin. 1984, 31, 703-705. (16) Yguerabide, Y.; Burton, M. J . Chem. Phys. 1962, 37, 1757. (17) Kawski, A.; Kukielski, J.; Kaminski, J. Z . Nuzurforsch., A: Phys., Phys. Chem., Kosmophys. 1979, 34A, 1066-1069. (18) Nakajima, A. Spectrochim. Acta, Parr A 1983, 34A, 912-915. (19) Patonay, G.; Rollie, M. E.; Warner, I. M. Anal. Chem. 1985, 57, 569-571. (20) Cline Love, L. J.; Weinberger, R. Spectrochim. Acta, Part E 1983, 38E, 1421-1433. (21) Cohen, Z . Org. Synrh. 1979, 59, 176-182.
0022-3654/88/2092-1052$01.50/00 1988 American Chemical Society
The Journal of Physical Chemistry, Vol. 92, No. 5, 1988 . 1053
PPO-y-CD Aggregates
/ ~
/ 1
I
I
I
1
I
250 260 270 280 290 300 310 320 330 340 350 360 370
inm)
(nm)
Figure 1. Formula of 2,5-diphenyloxazole (PPO). M fluorescence emission spectra of 25 nM PPO in 1 mM of a-,(3-, and y-CD. The order of the respective intensities may be significant.
Xex 3 4 5 nm
Figure 3. UV absorbance of PPO-CD complexes in water at room temperature. Solutions (0.01 M) of a-,b-, and y-CD had reacted with uncalibrated [PPO]. a and (3 yielded M-type absorption spectra, while y showed a mixed M and D spectrum.
Xex=313nm
p\
Xex 345nm
(nm) Figure 2. Fluoresence emission of concentrated (0.25 mM) PPO with CD at 0.01 M. The (3-CD complex always yielded M spectra. The y-CD complex yielded mixed M and D at A,, of 313 nm and pure D at A, of
345 nm. Professor L. Brand and Dr. J. Beechem.zz Dynamic light scattering was performed on a Malvern Instruments photon correlation spectrometer, equipped with a 4700M/SM system. The laser was 35 mW He-Ne. The software treated the data as if the scatterers were spherical and yielded a distribution of Stokes-Einstein radii. Results The main results of this work were (1) the self-assembly of extended molecular aggregates, held together by PPO-y-CD inclusion complexation, and (2) excimer PPO fluoresence and ‘dimer” absorption, exclusively connected with this mode of association. We first present the evidence for the existence of the aggregates and then report their properties in detail. The evidence is as follows: (1) visible turbidity, absent in an equimolar (10 mM) solution of y-CD with no PPO. This proved the essential role of PPO in holding the aggregates together. (2) Dynamic laser light scattering yielded a distribution of aggregate sizes in the micrometer range (Figure 7). The macromolecular size of the aggregates was thus demonstrated, even though the computation was in terms of spherical scatterers. (3) The large polarization of PPO excimer fluorescence (Figure 6) indicated that during the rather long lifetime of that species (16 ns), the emitters did not tumble. (22) Knutson, J. R.; Beechem, J. M.; Brand, L. Chem. Phys. Lett. 1983, 102, 501-507.
(nm) Figure 4. Aggregate formation as a function of [y-CD]. Fluorescence of 25 pM PPO with (a) 1, (b) 2, and (c) 10 mM of y-CD. D builds up at the expense of M.
Detailed Results. UV absorption (Figures 3 and 5) and fluorescence (Figures 1,2,4, and 8-10) spectra were of two types: (I) monomer (=M), observed in dilute PPO solutions in organic solvents,I0 in water solutions of cy- and 8-CD (Figure l), in dilute water solutions of y C D (Figure l), and in PPO crystals (Figure 8); (11) excimer (dimer = D) fluorescence, obtained only in the aggregates described here (Figures 2, 4, 9, and 10) and in PPO fluorescence in concentrated solutions, where it was discovered in the first D-type UV absorption (Figures 3 and 5 ) appeared only in the aggregates and not in organic solvents. D absorption was due to electronic transitions from ground state into a preexistent “excimer” state in the aggregate. In solution, “encounter excimers” were formed by molecules previously excited into a “monomer” state, so that only M absorption took place. Hence it was advisable to apply the name dimer rather than excimer to the appropriate aggregate spectra. Aggregate buildup was made possible only by our preparation technique. Solutions with increasing concentrations of T-CD were exposed to solid-adsorbed PPO. The resulting increase in complex concentration was indicated by the growth of the M-type spectra
Agbaria and Gill
1054 The Journal of Physical Chemistry, Vol. 92, No. 5, 1988 Xex = 3 4 0 n m
Xex = 3 1 3 n m
a
m K
0
m m
a
200
I
I
250
300
350
(nm) Figure 5. UV absorbance of PPO in solution with 10 mM y-CD. PPO concentrations are (a) 25 nM, (b) 250 nM, and (c) 0.25 mM. Part c is a mix of M and D absorption. This spectrum is strongly attenuated by scattering from aggregates, present only in this sample. PO LA RI Z A T I3h
c 300
F LUORESCE NC E 6
e259
9.1 cc
SPECTRA
EXCITATION
b
!
20
(nm) Figure 8. M emission from pure PPO powder.
X e x = 313 nm
Lex= 340nm
'
I 1
Figure 6. Polarized spectra of excitation (1, 3, 5) and emission (2,4,6) of fluorescence of PPO in 8-CD (1,2) and y C D (3-6).Emissions 2 and 4 were scanned at A,, = 313 nm. Emission 6 was scanned at A,, = 345 nm. Excitations 1 and 3 were scanned at ,A, = 370 nm, and excitation 5 was scanned at ,A, = 415 nm.
500
I
I
450
400
L
35
( n m ) Figure 9. Fluorescence emission of newly dried aggregate. X e x = 313
h e x = 345 nm
I
200
2000
Particle size distribution (nm)
Figure 7. Dynamic laser light scattering data, interpreted in terms of size distribution of spherical scatterers. In spite of the error in applying this model to filamentary scatterers, their macromolecular size is evident. The steepness of the smaller size slope is due to the log presentation. A mean aggregation number of 500 is estimated from this curve. (Figure 4). Since PPO was insoluble in water, the soluble species contributing t h e M spectra was assigned to small inclusion complexes, in which PPO molecules did not interact. W h e n 2 mL of 10 m M y C D (containing 2 X mol) reacted with 5 X lo-* mol of glass-adsorbed PPO,t h e rise of D spectra a t t h e expense of M spectra was started (Figure 4). Only then did other ag-
u 500
450
400
C
1
450
400
350
(nm) Figure 10. Emission of dried aggregate after 2 weeks of storage at room temperature. gregate properties begin to appear. T h e aforementioned threshold quantities were minimal a n d aggregate growth was augmented
PPO-y-CD Aggregates TABLE I: Fluorescence Anisotropy of Inclusion Complexes of PPO in CY-, &,and y - C P r A,, = 313 nm A,, = 345 nm AI = 370 nm AI = 420 nm no. CD mol wt l a 972.86 0.040 0.038 0.044 0.080 2 0 1135.01 0.100 0.217 3 7 1297.15 M type 4 7 1297.15 0.094 0.235 D type
’[CD] = 0.01 M in all samples. [PPO] was 25 pM in samples 1, 2, and 4 and 0.25 pM in sample 3. in vials containing more PPO or y-CD. Direct excitation of D absorption at 358 nm yielded nearly pure D fluorescence, while excitation at 316 nm always gave both M and D fluorescence at various intensity ratios (Figures 2 and 4). We could not distinguish between homogeneous and heterogeneous PPO populations. In the homogeneous case, all the molecules would overlap evenly, yet each excited molecule would emit either M or D at some probability ratio. Heterogeneity would apply to PPO subgroups, only some of which are dimeric. The spectral separation between M and D fluorescence excitation and emission spectra allowed independent lifetime and anisotropy measurements. A single photon correlation fluorometer was excited by N, microflash at 316 or 358 nm. The response to excitation at 316 nm consisted of two spectrally separated decays: a single exponential of C1 ns at 370 nm and a single exponential of 16 ns at 450 nm. This lifetime, longer than the 13.6-11s component reported J ~ indicated a common for PPO excimers in s ~ l u t i o n , ’ ~still mechanism. Excitation at 358 nm evoked a single exponential decay at 450 nm. The monoexponential decays indicated tg us that processes intervening between excitation at 316 nm and excimer emission were too fast to be detected by our equipment. On the other hand, absorption at 358 nm could not excite monomer emission. Both processes, neither of which gave rise to measurable excimer k i n e t i ~ s , ’ ~attested J~ to the stability of the aggregate. Sine-wave modulated flourometry data were obtained in terms of phase and modulation lifetimes. When excited at 345 nm, detected at 420 nm, and modulated at 18 MHz, r(mod) = 16.6 ns was obtained, in good agreement with the one listed above. Steady fluorescence anisotropy measurements were applied separately to M and D spectra (Figure 6, Table I). The Perrin-Weber formula ro/ ( r ) = 1 + T / ( Pcould be utilized to estimate a lower bound of aggregate size. Not knowing ro in frozen PPO, we adopted 0.3, the ro value for POPOP.’’ The measured ( r ) of the D fluorescence was near 0.2 (Figure 6). With 7 = 16 ns, the rotational correlation time cp came out to be 32 ns. According to the empirical rule of 3 kdalton/ns, the aggregate molecular weight was 96 000. Taking the combined molecular weight of one y-CD and two PPO as 1500, we got an aggregation number of -60. Solid PPO powder showed pure M fluorescence (Figure 8). A practical conclusion was that the spectra of suspended PPO crystallites, if any, would not be confused with those of the aggregates.
The Journal of Physical Chemistry, Vol. 92, No. 5, 1988 1055 We attempted to dry the aggregates and check their yield of D fluorescence after storage. The strong D component of the newly dried aggregate (Figure 9) left some trace after 2 weeks (Figure 10). Without pursuing the subject, we feel that the aggregates were destabilized by loss of water.
Discussion The reported results, when combined with CPK molecular modeling, suggest a pattern of aggregate structure. The basic assumption is that the heterocyclic middle ring of PPO is hydrophilic, probably hydrated, and perhaps hydrogen bonded to water. Therefore, only a phenyl ring of PPO would penetrate a C D cavity, while the middle ring would stay in the water phase. Moreover, PPO molecules would not stack when in water and thus would resist the formation of excimers. The soluble complex giving rise to M spectra is assumed to be a 2:l complex in “contran configuration. Two PPO molecules would partly penetrate the opposite ends of a CD tunnel (a shorthand notation for this complex would be PCP). PPO molecules are short and, being out of touch inside the tunnel, yield M spectra. The “contra” complex (PCP) is more stable than the “co-” complex (CPP), which would have yielded D spectra (the opposite applies to pyrene6). At high concentration of PCP, these units would associate and form coaxial arrays of y-CD beads, linked by overlapping pairs of PPO molecules. (The notation would be (PCP), or (PCPPCPP...CP.) Thus, PPO pairs are compelled to overlap only when they are accommodated between adjacent y-CD beads. The short lengths of the PPO links would bring the y-CD beads to line up rather tightly. The overlap geometry of the PPO pair is not known and sheer congruence is only one of several possibilities. Some of those may be eliminated by the spectroscopic evidence of red-shifted D absorption and emission relative to those of M. We would defer this reasoning to a time when structural data become available. a- and 0-CD would also give rise to PCP complexes. As the cavity aperture is too small to accommodate a pair of PPO, the units would not associate. One may still conceive of CY- or p-CD aggregates, linked by single PPO (namely, PCPCPC...). The prerequisite would be a high concentration of the 1:l (PC) complex and the absence of the PCP complex. The nonexistence of such aggregates proves by contradiction that PCP complexes of CY- and p-CD are more stable than PC. The proposed model is feasible, yet more supporting evidence is needed. The aggregate would be a thin filament of 1.7 nm diameter and a length of N X 0.7 nm, where N is the aggregation number, reaching the hundreds. The aggregates are expected to have anisotropic properties, which presently are under study. The total absence of D spectra in PPO powder indicates the absence of favorable overlap in the unit cell.23 Acknowledgment. We are indebted to Professor N. J. Turro, Professor J. Bernstein, Dr. A. H. Parola, and Mr. Y. Cohen for much advice and help. Registry No. PPO-a-CD ( l : l ) , 111743-27-2; PPO-fi-CD ( l : l ) , 111743-28-3; PPO-7-CD (Irl), 111743-29-4; PPO-Y-CD (2:1), 11 1743-30-7; PPO, 92-71-7. (23) Stevens, B. Spectrochim. Acta 1962, 18, 439.
